Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition.

INTRODUCTION: Triple-negative breast cancer (TNBC) is aggressive and lacks targeted therapies. Phosphatidylinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are frequently activated in TNBC patient tumors at the genome, gene expression and protein levels, and mTOR inhibitors have been shown to inhibit growth in TNBC cell lines. We describe a panel of patient-derived xenografts representing multiple TNBC subtypes and use them to test preclinical drug efficacy of two mTOR inhibitors, sirolimus (rapamycin) and temsirolimus (CCI-779). METHODS: We generated a panel of seven patient-derived orthotopic xenografts from six primary TNBC tumors and one metastasis. Patient tumors and corresponding xenografts were compared by histology, immunohistochemistry, array comparative genomic hybridization (aCGH) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) sequencing; TNBC subtypes were determined. Using a previously published logistic regression approach, we generated a rapamycin response signature from Connectivity Map gene expression data and used it to predict rapamycin sensitivity in 1,401 human breast cancers of different intrinsic subtypes, prompting in vivo testing of mTOR inhibitors and doxorubicin in our TNBC xenografts. RESULTS: Patient-derived xenografts recapitulated histology, biomarker expression and global genomic features of patient tumors. Two primary tumors had PIK3CA coding mutations, and five of six primary tumors showed flanking intron single nucleotide polymorphisms (SNPs) with conservation of sequence variations between primary tumors and xenografts, even on subsequent xenograft passages. Gene expression profiling showed that our models represent at least four of six TNBC subtypes. The rapamycin response signature predicted sensitivity for 94% of basal-like breast cancers in a large dataset. Drug testing of mTOR inhibitors in our xenografts showed 77 to 99% growth inhibition, significantly more than doxorubicin; protein phosphorylation studies indicated constitutive activation of the mTOR pathway that decreased with treatment. However, no tumor was completely eradicated. CONCLUSIONS: A panel of patient-derived xenograft models covering a spectrum of TNBC subtypes was generated that histologically and genomically matched original patient tumors. Consistent with in silico predictions, mTOR inhibitor testing in our TNBC xenografts showed significant tumor growth inhibition in all, suggesting that mTOR inhibitors can be effective in TNBC, but will require use with additional therapies, warranting investigation of optimal drug combinations.

ß-Catenin transcriptional activity is minimal in canine osteosarcoma and its targeted inhibition results in minimal changes to cell line behaviour.

Canine osteosarcoma (OS) is an aggressive malignancy associated with poor outcomes. Therapeutic improvements are likely to develop from an improved understanding of signalling pathways contributing to OS development and progression. The Wnt signalling pathway is of interest for its role in osteoblast differentiation, its dysregulation in numerous cancer types, and the relative frequency of cytoplasmic accumulation of ß-catenin in canine OS. This study aimed to determine the biological impact of inhibiting canonical Wnt signalling in canine OS, by utilizing either ß-catenin siRNA or a dominant-negative T-cell factor (TCF) construct. There were no consistent, significant changes in cell line behaviour with either method compared to parental cell lines. Interestingly, ß-catenin transcriptional activity was three-fold higher in normal canine primary osteoblasts compared to canine OS cell lines. These results suggest canonical Wnt signalling is minimally active in canine OS and its targeted inhibition is not a relevant therapeutic strategy.

Wnt/ß-catenin expression does not correlate with serum alkaline phosphatase concentration in canine osteosarcoma patients.

Osteosarcoma is an aggressive malignancy of the bone and an increase in serum alkaline phosphatase concentration has clinical prognostic value in both humans and canines. Increased serum alkaline phosphatase concentration at the time of diagnosis has been associated with poorer outcomes for osteosarcoma patients. The biology underlying this negative prognostic factor is poorly understood. Given that activation of the Wnt signaling pathway has been associated with alkaline phosphatase expression in osteoblasts, we hypothesized that the Wnt/ß-catenin signaling pathway would be differentially activated in osteosarcoma tissue based on serum ALP status. Archived canine osteosarcoma samples and primary canine osteosarcoma cell lines were used to evaluate the status of Wnt/ß-catenin signaling pathway activity through immunohistochemical staining, western immunoblot analyses, quantitative reverse-transcription polymerase chain reaction, and a Wnt-responsive promoter activity assay. We found no significant difference in ß-catenin expression or activation between OSA populations differing in serum ALP concentration. Pathway activity was mildly increased in the primary OSA cell line generated from a patient with increased serum ALP compared to the normal serum ALP OSA cell line. Further investigation into the mechanisms underlying differences in serum ALP concentration is necessary to improve our understanding of the biological implications of this negative prognostic indicator.

Runx1 directly promotes proliferation of hair follicle stem cells and epithelial tumor formation in mouse skin.

Runx1/AML1 is a transcription factor implicated in tissue stem cell regulation and belongs to the small Runx family of cancer genes. In the hair follicle (HF), Runx1 epithelial deletion in morphogenesis impairs normal adult hair homeostasis (cycle) and blocks adult hair follicle stem cells (HFSCs) in quiescence. Here, we show that these effects are overcome later in adulthood. By deleting Runx1 after the end of morphogenesis, we demonstrate its direct role in promoting anagen onset and HFSC proliferation. Runx1 deletion resulted in cyclin-dependent kinase inhibitor Cdkn1a (p21) upregulation. Interfering with Runx1 function in cultured HFSCs impaired their proliferation and normal G(0)/G1 and G(1)/S cell cycle progression. The proliferation defect could be rescued by Runx1 readdition or by p21 deletion. Chemically induced skin tumorigenesis in mice turned on broad Runx1 expression in regions of the skin epithelium, papillomas, and squamous cell carcinomas. In addition, it revealed reduced rates of tumor formation in the absence of Runx1 that were accompanied by decreased epithelial levels of phospho-Stat3. Runx1 protein expression was similar in normal human and mouse hair cycles. We propose that Runx1 may act as a skin oncogene by directly promoting proliferation of the epithelial cells.